Tumoricidal and antimicrobial compositions and methods

ABSTRACT

Provided herein are pharmaceutical compositions comprising tumoricidal and/or antimicrobial components isolated from the supernatant of NK-92 cell medium and methods of using the compositions for killing cancer cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 17/539,155, filed Nov. 30, 2021, which is a continuation of U.S.application Ser. No. 16/598,418, filed Oct. 10, 2019, which is acontinuation application of U.S. application Ser. No. 16/289,494, filledFeb. 28, 2019, which is a divisional application of U.S. applicationSer. No. 15/032,518, filed Apr. 27, 2016, which is a 35 USC § 371national stage entry of International Application No. PCT/US2014/062695,filed Oct. 28, 2014, which claims the benefit of U.S. ProvisionalApplication No. 61/899,066, filed Nov. 1, 2013, all of which areincorporated by reference herein in their entirety for all purposes.

FIELD OF THE INVENTION

This invention relates generally pharmaceutical compositions havingtumordical and/or antimicrobial properties and methods of using thesecompositions for killing cancer and/or microbes.

STATE OF THE ART

Skin cancer is the leading type of cancer in humans as well as manydomesticated animals. The occurrence of skin cancer and melanomas areincreasing significantly due to a variety of factors including exposureof unprotected skin to UV rays, such as those found in sunlight ortanning beds. When diagnosed at their incipient stage, these cancers arereadily treatable by excision of the cancer and surrounding tissue.However, as in any surgical procedure, there remains the possibilitythat aberrant cancer cells may be retained at the site of the incisionthereby causing recurrence of the cancer. This is particularly the casefor melanomas, and the failure to remove all of the tumor can result inmetastasis accompanied by high levels of morbidity.

There are numerous aggressive protocols that can be taken to minimizethe risk of recurrence including conventional therapeutic protocols aswell as numerous checkups with the attending clinician. Notwithstandingsuch protocols, melanomas tend to be very aggressive, and it is reportedthat melanoma has a 2% to 65% likelihood of recurrence within 5 years,depending on the stage of the cancer at treatment. See, e.g.,www.aimatmelanoma.org/en/aim-for-answers/moving-on-after-treatment/follow-up-by-stage.html.

Animals are also prone to skin cancers, especially melanoma, squamouscell carcinoma, and mast cell tumors. Skin cancers are especiallyprevalent in animals that spend a lot of time in the sun.

Infections are a common problem throughout the world. Many infectionsare caused by bacteria, fungi, and other microbes. Although currenttreatment for such infections relies heavily on antibiotics andantimicrobial drugs, an increasing number of bacterial infections arefound to be resistant to at least some antibiotics. The CDC reports thatover two million Americans are infected by antibiotic-resistant microbesevery year, resulting in more than 23,000 deaths. See, e.g.,www.medicalnewstoday.com/articles/266182.php. Skin and soft tissueinfections represent the third most common diagnosis in emergency caresettings, and an estimated 7% to 10% of all hospitalized patients have askin or soft tissue infection. Ki and Rotstein, Can. J. Infect. Dis.Med. Microbiol. 2008 March; 19(2): 173-184. Other common infectionsinclude systemic infections, respiratory infections, ear infections,gastrointestinal infections, and urinary tract infections. Infectionsare similarly common in domesticated animals and can be difficult totreat. Viral infections are also common, and few treatments areavailable for treating such infections.

Accordingly, there remains a need for tumorcidal compositions useful inlysing cancer cells, especially those related to dermal and subdermalcancers. There also remains a need for novel antimicrobial compositionsuseful in treating infection.

SUMMARY OF THE INVENTION

Certain cells of the immune system have cytotoxic activity againstparticular target cells. Natural killer (NK) cells, generallyrepresenting about 10-15% of circulating lymphocytes, bind and killtarget cells, including virus-infected cells and many malignant cells,nonspecifically with regard to antigen and without prior immunesensitization. Herberman et al., Science 214:24 (1981). Killing oftarget cells occurs by inducing cell lysis. NK cells have been shown tobe effective in both ex vivo therapy and in vivo treatment in patientswith advanced cancer. However, endogenous NK cells (i.e., those that areharvested from a donor or from the patient) remain difficult to workwith and to apply in immunotherapy. It is difficult to expand NK cellsex vivo such that they maintain their tumor-targeting, tumoricidal, andviricidal capabilities in vivo, a major obstacle to their clinical usein adoptive cell immunotherapy. Melder, et al., Cancer Research48:3461-3469 (1988); Stephen, et al., Leuk. Lymphoma 377-399 (1992);Rosenberg, et al., New Engl. J. Med. 316:889-897 (1987). Further,preparations of endogenous NK cells include T cells and/or other immuneeffector cells that must be removed if the NK cells are used to treat apatient unrelated to the donor.

The NK-92 cell line is a unique cell line that was discovered toproliferate in the presence of interleukin 2 (IL-2). Gong et al.,Leukemia 8:652-658 (1994). Unlike NK cells, NK-92 is a cytolytic cancercell line which was discovered in the blood of a subject suffering froma non-Hodgkins lymphoma and then immortalized ex vivo. This cell linehas high cytolytic activity against a variety of cancers. The NK-92 cellline is a homogeneous NK cell population as it relates to its lysingactivity. Phase I clinical trials have confirmed its safety profile, andanti-tumor responses in certain patients with advanced cancer have beenobserved.

Endogenous NK cells are significantly different from NK-92 cells, inlarge part because of their distinct origins: NK-92 is a cancer-derivedcell line, whereas endogenous NK cells are harvested from a donor (orthe patient) and processed for infusion into a patient. Endogenous NKcell preparations are heterogeneous cell populations, whereas NK-92cells are a clonal cell line that are homogenous in that they allexhibit lyzing activity. NK-92 cells readily proliferate in culturewhile maintaining cytotoxicity, whereas endogenous NK cells do not.

Cells, including NK cells, release a variety of components into themedium in which they are grown. Examples of such components areproteins, exosomes, and microvesicles. Exosomes are nanovesicles (up to100 nm) that are released by a variety of normal and tumor cells.Microvesicles are similar to exosomes but larger in size (greater than100 nm). Exosomes and microvesicles can be detected and isolated fromcell culture supernatants and from body fluids (e.g., blood).

Exosomes isolated from the cell culture supernatant of endogenous NKcells contain proteins including CD56, perforin, FasL, and Rab5B.Lugini, et al. J Immunol. (2012) 189, 2833-2842. However, exosomes andother factors derived from endogenous NK cells are highly variable, bothwith regard to the amount of exosomes recovered and the proteinscontained therein. Id. at 2839. Without being bound by theory, it isbelieved that this variability arises because endogenous NK cells areharvested from individual donors, and also because endogenous NK cellscomprise a heterogeneous population of cells. Differences in conditionssuch as cell culture conditions, purification methods, initial cellpopulations, and the ratios of cell types in culture can all lead tovariability in the composition and amount of exosomes that can bepurified from endogenous NK cells. For example, certain cell populationswithin the NK cell preparations may be selected for or against by thepurification method and/or culture conditions used.

The problems with variability that are associated with endogenous NKcells do not apply to the NK-92 cell line. Because it is a cell line,large numbers of cells can be cultured and propagated for extendedperiods of time. These cultures are homogeneous cell populations thatprovide consistent, reproducible exosome and/or microvesiclepreparations. The exosomes and/or microvesicles secreted by NK-92 cellscontain proteins that are contemplated to have tumoricidal properties,for example are cytotoxic and/or cytolytic. Other components may beisolated from the medium containing NK-92 cells, including componentswith antimicrobial properties, immunomodulatory properties, etc.

On the other hand, NK-92 cells are a cancer cell line. Some cancer cellshave been shown to release exosomes containing factors that in somesituations contribute to tumor growth, for example micro RNAs. Manycancer cells release exosomes and other factors that are distinct fromthose released by their non-malignant counterparts. For example, NK-92cells release factors with anti-microbial properties, a characteristicnot observed in endogenous NK cells. In one embodiment, the exosomesand/or microvesicles obtained from NK-92 cells are incubated in asuitable solution, such as PBS or isotonic saline, to extract suchfactors prior to their use. This incubation period is contemplated tosignificantly reduce or eliminate those factors that contribute to tumorgrowth.

One aspect of the invention provides pharmaceutical compositionscomprising one or more components obtained from the supernatant of NK-92cell medium. The components may be tumoricidal and/or anti-microbial.Components may also have immunomodulatory properties. These componentsare preferably exosomes and/or microvesicles isolated from thesupernatant. In one embodiment, the exosomes and/or microvesicles arecytotoxic. In one embodiment, the exosomes and/or microvesicles have theability to lyse cancer cells. In one embodiment, the exosomes and/ormicrovesicles are antimicrobial. In one embodiment, the exosomes and/ormicrovesicles are antiviral. In one embodiment, the exosomes and/ormicrovesicles are antibacterial. In one embodiment, the exosomes and/ormicrovesicles are immunomodulatory.

In a preferred embodiment, the pharmaceutical composition does notcomprise living cells. In some embodiments, the pharmaceuticalcomposition is an injectable composition. In some embodiments, it iscontemplated that the injection will provide systemic immunomodulatoryactivity.

Subpopulations of NK-92 cells have been observed in culture and can beseparated. Subpopulations may differ in terms of expression of cellsurface markers, protein expression, etc. In one aspect of theinvention, one or more subpopulations of NK-92 cells are isolated priorto obtaining the one or more components from the supernatant of NK-92cell medium. Isolation of a defined subpopulation or subpopulationsallows for consistently defined exosomes and/or microvesicles, and inparticular exosomes and/or microvesicles that exhibit less or notumoricidal properties.

In some embodiments, the pharmaceutical composition provided herein isapplied to a part of the body after surgery in order to kill remainingcancer cells and/or to reduce the possibility of recurrence of thecancer. In some embodiments, the pharmaceutical composition isformulated for topical use. In some embodiments, the pharmaceuticalcomposition is formulated to be applied subdermally. In someembodiments, the pharmaceutical composition comprises a phase-transitionpoloxamer.

In some embodiments, the pharmaceutical composition is an injectibleform which comprises a phase transition polymer such that thecomposition is injected as a liquid and phase transitions to a gel inthe body (e.g., at body temperature) thereby providing a drug depot.

Another aspect of the invention provides pharmaceutical compositionscomprising anti-microbial (including anti-bacterial, anti-fungal and/oranti-viral) components obtained from the supernatant of NK-92 cellmedium. In some embodiments, the anti-microbial components comprisetumoricidal components. In some embodiments, the anti-microbialcomponents are exosomes and/or microvesicles isolated from thesupernatant. In a preferred embodiment, the anti-microbial componentscomprise microvesicles isolated from the supernatant.

Another aspect of the invention provides methods of lysing cancer cellscomprising administering to a human or animal patient in need thereof anantitumor or cytotoxic component isolated from the supernatant of thegrowth medium of NK-92 cells. In one embodiment, the component isinjected into a tumor (e.g., a solid tumor). In one embodiment, thecomponent is injected into the area around or near a tumor. In oneembodiment, the component is applied topically to a tumor (e.g., skincancer), In one embodiment, the component is administered systemically.

In some embodiments, the cancer is a carcinoma, lymphoma, sarcoma,melanoma, astrocytoma, mesothelioma cells, ovarian carcinoma, coloncarcinoma, pancreatic carcinoma, esophageal carcinoma, stomachcarcinoma, lung carcinoma, urinary carcinoma, bladder carcinoma, breastcancer, gastric cancer, leukemia, lung cancer, colon cancer, centralnervous system cancer, ovarian cancer, cervical cancer, renal cancer, orprostate cancer. In a preferred embodiment, the cancer is skin cancer.

These and other aspects of the invention will be set forth in detailsbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of NK-92 cells on Cryptococcus neoformansgrowth.

FIG. 2 shows the same data as in FIG. 1, expressed as a percent of C.neoformans growth.

FIG. 3A is a picture of a Western blot analyzing the protein content ofexosomes/microvesicles (EV/MV) isolated from NK-92 cells under a varietyof culture conditions (lanes 4-7). NK-92 cells (lane 3) are used as apositive control. MCF-7 cells (lane 1) and exosomes (EV, lane 2) arepositive controls for tubulin and/or Rab5B, and negative controls forcytolytic proteins.

FIG. 3B is a picture of a Western blot analyzing the presence of markersfor nuclear membrane (nucleoporin), mitochondria (prohibitin) andexosomes (Rab5B) in NK-92 cells and exosome/microvesicle preparation.

FIG. 4 shows the cytolytic activity of the NK-92 exosome/microvesiclepreparation against Jurkat cells in a propidium iodide assay.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before the present articles and methods are disclosed and described, itis to be understood that the aspects described below are not limited tospecific compositions, preparation methods, or uses as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular aspects only and isnot intended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a cytokine” includes mixtures of two or more cytokines,and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. For example, a composition consistingessentially of the elements as defined herein would not exclude otherelements that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanexcluding more than trace amount of other ingredients and substantialmethod steps recited. Embodiments defined by each of these transitionterms are within the scope of this invention.

The term “patient” as used herein is any vertebrate organism includingbut not limited to mammalian patients such as humans, farm animals,domesticated pets and the like. In a preferred embodiment, the patientis a human.

The term “about” when used before a numerical value indicates that thevalue may vary within a reasonable range, such as ±5%, ±1%, and ±0.2%.

The term “endogenous NK cells” is used to refer to NK cells derived froma donor (or the patient), as distinguished from the NK-92 cell line. ANK cell is a cell of the immune system that kills target cells in theabsence of a specific antigenic stimulus, and without restrictionaccording to MEW class. Endogenous NK cells are generally heterogeneouspopulations of cells within which NK cells have been enriched.Endogenous NK cells may be intended for autologous or allogeneictreatment of a patient.

The term “NK-92 cell” includes both wild type NK-92 cells and modifiedNK-92 cells. NK-92 cells were found to be more cytotoxic to tumor andinfected cell types than are NK cells.

The term “wild type NK-92 cell” refers to an NK cell line, NK-92,originally obtained from a patient having non-Hodgkin's lymphoma andimmortalized ex vivo. NK-92 cells are available from American TypeCulture Collection as Deposit No. CRL-2407, and described in, e.g., U.S.Pat. No. 7,618,817, which is incorporated by reference in its entirety.

The term “modified NK-92 cell” refers to an NK-92 cell which has beenfurther treated to endow it with properties not found in the wild typeNK-92 cell from which it is derived. Such treatments include, forexample, physical treatments, chemical and/or biological treatments, andthe like. The treatments confer properties upon the modified NK-92 cellsthat render them more advantageous for the desired purposes. Examples ofmodified NK-92 cells are described in, e.g., U.S. Pat. Nos. 7,618,817;8,034,332; and 8,313,943, all of which are incorporated herein byreference in their entireties.

The term “extracellular vesicle” encompasses both exosomes andmicrovesicles, as well as any other vesicle secreted by a cell (e.g., aNK-92 cell) into the medium. Generally, exosomes are nanosomes that areless than about 100 nanometers (nm) in diameter. Microvesicles have adiameter of about 100 nm or larger.

As used to describe the present invention, “cancer”, “tumor”, and“malignancy” all relate equivalently to a hyperplasia of a tissue ororgan. If the tissue is a part of the lymphatic or immune system,malignant cells may include non-solid tumors of circulating cells.Malignancies of other tissues or organs may produce solid tumors. Ingeneral, the methods of the present invention may be used in thetreatment of lymphatic cells, circulating immune cells, and solidtumors.

The term “tumoricidal component” refers to components that treatcancerous tumors and/or cancer cells. Treatment of tumors encompassesreducing or eliminating the tumor, killing cancer cells, and/orinhibiting the growth, proliferation, and/or metastasis of cancer cells.Preferably, the tumor cells are killed, for example by cytolysis.

The term “anti-microbial component” refers to components that treat orprevent infection by microbes. Microbes include bacteria, fungi, molds,viruses, etc. Accordingly, anti-microbial also refers to anti-viralcomponents, anti-bacterial components, anti-fungal components, and thelike.

As used to describe the present invention, the terms “cytotoxic” and“cytolytic”, when used to describe the activity of effector cells suchas NK cells, are intended to be synonymous. In general, cytotoxicactivity relates to killing of target cells by any of a variety ofbiological, biochemical, or biophysical mechanisms. Cytolysis refersmore specifically to activity in which the effector lyses the plasmamembrane of the target cell, thereby destroying its physical integrity.This results in the killing of the target cell. Without wishing to bebound by theory, it is believed that the cytotoxic effect of NK cells isdue to cytolysis.

The term “growth medium” or “medium” as used herein are intended to besynonomous. In general, the terms refer to any medium or aqueoussolution in which the NK-92 cells may be grown or placed and into whichthe NK-92 cells release exosomes, microvesicles, and/or other activecomponents. Medium may comprise growth mediums (e.g., commerciallyavailable medium) such as X-VIVO 10 or RPMI. Alternatively, medium maycomprise PBS or other aqueous solution.

As used herein, “treatment,” “treating,” and “treat” are defined asacting upon a disease, disorder, or condition with an agent to reduce orameliorate harmful or any other undesired effects of the disease,disorder, or condition and/or its symptoms. “Treatment,” as used herein,covers the treatment of a patient, and includes: (a) reducing the riskof occurrence of the condition in a patient determined to be predisposedto the condition but not yet diagnosed as having the condition, (b)impeding the development of the condition, and/or (c) relieving thecondition, i.e., causing regression of the condition and/or relievingone or more symptoms of the condition. “Treating” or “treatment of” acondition or patient refers to taking steps to obtain beneficial ordesired results, including clinical results such as the reduction ofsymptoms. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to: reducing the size or metastaticpotential of a tumor; killing tumor cells; or reducing the severity ofinfection by an infections agent (microbe), e.g., by reducing one ormore symptoms, reducing the length of time of infection, etc.

Components Isolated from the Supernatant of a Medium of NK-92 Cells

NK-92 cells can be expanded, modified and/or maintained in culturemedium. Any acceptable culture conditions may be used. In oneembodiment, NK-92 cells are cultured in enriched alpha minimum essentialmedium (MEM; Sigma Chemical Co., St. Louis, Mo.) supplemented with fetalcalf serum (for example, at 12.5%; Sigma Chemical Co., St. Louis, Mo.),and/or horse serum (for example, at 12.5%; Sigma Chemical Co., St.Louis, Mo.). In another embodiment, the NK-92 cells are cultured inXVivo 10 medium supplemented with human serum, human plasma, or humanserum albumin (for example, at 5%). In a preferred embodiment, theserum, plasma, or serum albumin is exosome-depleted prior to culture ofthe NK-92 cells.

The medium optionally is supplemented with other nutrients, cytokines,and/or growth factors, for example interleukin 2 (IL-2), L-asparagine,L-glutamine, and/or L-serine. NK-92 cells, when in the medium, canrelease components such as proteins (e.g., cytokines), microvesicles,and/or exosomes to the medium. After the cells are isolated from themedium, for example, by centrifugation, the components released from thecells remain in the supernatant.

Another suitable medium employed includes X-VIVO 10 medium, 5% humanserum AB, 36 μM L-asparagine, 450 μM L-glutamine, 324 μM L-serine, and500 IU of IL-2.

In some embodiments, the medium is serum-free medium, PBS, or otheraqueous solution, for example Ringer's solution, dextrose solution,Hank's solution, and other aqueous physiologically balanced saltsolutions. Without being bound by theory, some components of growthmedium (e.g., serum) may contain exosomes, microvesicles, and/or othercomponents that are unrelated to NK-92 cells. Accordingly, it may bebeneficial to maintain the NK-92 cells in serum-free medium or otheraqueous solution for a period of time before isolating exosomes,microvesicles, or other components from the medium. Alternatively, theNK-92 cells may be cultured in exosome-depleted or exosome-free serum orserum alternative.

In some embodiments, one or more stimulatory agents are added to themedium. Without being bound by theory, it is believed that stimulationof NK-92 cells with such agents may result in more consistent, robust,and/or reproducible release of components, including exosomes and/ormicrovesicles. Stimulatory agents include, for example, cytokines orpharmaceutical stimulators. In one embodiment, the stimulatory agent isIL-15. In one embodiment, the stimulatory agent is interferon gamma.

In some embodiments, the one or more tumoricidal and/or antimicrobialcomponents comprise exosomes isolated from the supernatant. In someembodiments, the one or more tumoricidal and/or antimicrobial componentscomprise microvesicles isolated from the supernatant. In someembodiments, the one or more tumoricidal and/or antimicrobial componentscomprise exosomes and microvesicles isolated from the supernatant.

Exosomes are nanovesicles excreted from the cells, having a diameter upto about 100 nm. In some embodiments, they have a diameter of about 30to about 100 nm. Microvesicles are non-cellular and have a diameter ofgreater than about 100 nm and preferably are less than about 1.5microns.

Exosomes and/or microvesicles can be isolated from medium by a number ofmethods. One method is by ultracentrifugation. Other methods includecommercially available exosome isolation kits (e.g., Total ExosomesIsolation kit [Life Technologies], Exo-spin™ Exosome Purification Kit[Cell Guidance Systems], or PureExo® Exosome Isolation Kit [101 Bio]);commercially available instruments, such as Dynabeads® HumanCD63-specific purification system or Dynabeads® Streptavidinpurification system (available from Life Technologies Corporation);filtration; or differential centrifugation methods (e.g., thosedescribed in S. Rani et al., Methods Mol Biol., 784:181-95 (2011)). Thepresence, size, and purity, etc. of exosomes and/or microvesicles can becharacterized by methods, such as Western blotting, transmissionelectron microscopy, flow cytometry, atomic force microscopy,nanoparticle tracking analysis, Raman microspectroscopy, resistive pulsesensing, and transmission electron microscopy.

In some embodiments, the NK-92 cells comprise wild type NK-92 cells.

In some embodiments, the NK-92 cells comprise modified NK-92 cells.NK-92 cells can be modified by methods known in the art, such as thosedescribed in U.S. Pat. No. 7,618,817, which is incorporated herein byreference in its entirety. For example, the NK-92 cells can be modifiedto express a Fc receptor on a surface of the cell. The Fc receptor canbe an activating Fcγ receptor, CD16 (FcγRIII-A), FCγRI (CD64), FCγRII(CD32), FCγRIII, FcRn, Fcα and Fcε, etc. The Fc receptors can be of anybinding affinity for their ligands, or fragments of their ligands,including low- and high-binding affinity forms. The NK-92 cells can befurther modified to express one or more associated accessory signalingpolypeptides, such as FcεRI-γ or TCR-ζ, cytokines, or fragments thereof.

In some embodiments, the NK-92 cells comprise NK-92 cells modified toexpress Fc receptors. In some embodiments, the NK-92 cells compriseNK-92 cells modified to express FcγRIII-A, FCγRI, FCγRII, FCγRIII, FcRn,Fcα or Fcε, or a combination thereof. In some embodiments, the NK-92cells comprise NK-92 cells modified to express one or more chimericantigen receptors. In some embodiments, the NK-92 cells comprisemodified NK-92 cells available from American Type Culture Collection asDeposit No. PTA-8836, PTA-6967, PTA-8837 or PTA-6672, or a combinationthereof. In some embodiments, the NK-92 cells comprise NK-92-CD16,NK-92-CD16-γ, or NK-92-CD16-ζ, or a combination thereof.

In some embodiments, the NK-92 cells comprise NK-92 cells modified toexpress a cytokine. In some embodiments, the NK-92 cells comprise NK-92cells modified to express a cytokine that promotes growth of the cellsand/or a cytokine receptor. In some embodiments, the NK-92 cellscomprise NK-92 cells modified to express IL-2 and/or IL-2 receptor. Insome embodiments, the NK-92 cells comprise NK-92 cells modified toexpress IL-15, IL-18, or IL-21, or a receptor thereof. In someembodiments, the NK-92 cells comprise modified NK-92 cells availablefrom American Type Culture Collection as Deposit No. CRL-2408 orCRL-2409, or a combination thereof. In some embodiments, the NK-92 cellscomprise NK-92MI, NK-92CI, or a combination thereof.

Without being bound by theory, it is believed that exosomes and/ormicrovesicles from modified NK-92 cells will be distinct from those fromwild type NK-92 cells. For example, exosomes and/or microvesiclesfrommodified NK-92 cells may contain different receptors and/or otherproteins (e.g., cytolytic enzymes), based on the modification(s) of thecells. Exosomes and/or microvesicles from modified NK-92 cells may alsocontain different amounts or relative amounts of some receptors and/orother proteins.

Pharmaceutical Compositions

In one aspect, provided is a pharmaceutical composition useful forkilling cancer cells in a warm-blooded animal, which compositioncomprises a pharmaceutically acceptable carrier and one or morecomponents isolated from the supernatant of a growth medium of NK-92cells.

In some embodiments, the pharmaceutical composition is liquid at roomtemperature and a gel when applied to the patient. In some embodiments,the pharmaceutically acceptable carrier comprises a poloxamer.

In another aspect, provided is a pharmaceutical composition comprisingexosomes and/or microvesicles isolated from the supernatant of a growthmedium of NK-92 cells and a sterile aqueous carrier.

In another aspect, provided is a kit comprising a first pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier andexosomes and/or microvesicles isolated from the supernatant of a growthmedium of NK-92 cells, and a second pharmaceutical compositioncomprising a gel, wherein the first pharmaceutical composition and thesecond pharmaceutical composition are topical formulations. In someembodiments, the second pharmaceutical composition a poloxamer. In someembodiments, the second pharmaceutical composition comprises exosomesand/or microvesiclesisolated from the supernatant of a growth medium ofNK-92 cells. In a preferred embodiment, the first and/or secondpharmaceutical component comprises microvesicles.

In some embodiments, the first pharmaceutical composition comprises aliquid and the second pharmaceutical composition comprises a gel,poloxamer, or composition that is a liquid at room temperature and a gelat body temperature. The liquid is applied to the treatment area first,and the gel is applied over the liquid. Without being bound by theory,it is believed that the liquid formulation provides rapid treatment ofthe area, while the gel maintains the liquid at the site of application.In some embodiments, the gel comprises exosomes and/or microvesiclesisolated from the supernatant of a growth medium of NK-92 cells and asterile aqueous carrier. Without being bound by theory, it is believedthat the gel will provide slower release of exosomes and/ormicrovesicles to the treatment area, thus providing for sustainedrelease and treatment of the affected area.

In some embodiments, the pharmaceutical composition further comprisesone or more cytokines.

The tumoricidal and/or antimicrobial component(s) may be administered inconjunction with a cytokine such as IFN-γ, TGF-β, IL-4, IL-10, IL-13,IL-2, etc., in order to maintain the functional effectiveness of thecomposition comprising the tumoricidal and/or antimicrobialcomponent(s). The term “in conjunction” indicates that the cytokine maybe administered shortly prior to administration of the compositioncomprising the component, or it may be given simultaneously with thecomposition comprising the component, or shortly after the compositioncomprising the tumoricidal component has been administered. The cytokinemay also be given at two such times, or at all three times with respectto the time of administering the composition comprising the tumoricidalcomponent. In some embodiments, the cytokine and the component areadministered in a single composition.

In some embodiments, the one or more cytokines includes at least IL-2.

The pharmaceutical composition can be in a variety of formulationssuitable for oral, topical, transdermal, rectal, inhalation, orparenteral (intravenous, intramuscular, or intraperitoneal)administration, and the like. The pharmaceutical composition can be in aformulation suitable for injection into a tumor or at or around a tumorsite. In one embodiment, the pharmaceutical composition is injected orapplied to a tumor site after surgery to remove all or most of thetumor.

As used herein “pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient” includes any and all solvents,dispersive agents or media, coating(s), antimicrobial agents,iso/hypo/hypertonic agents, absorption-modifying agents, and the like,suitable for pharmaceutical use and compatible with the tumoricidaland/or antimicrobial components. Moreover, other or supplementary activeingredients can also be incorporated into the final composition.

The pharmaceutical compositions described herein can be administered ina number of ways depending on whether local or systemic treatment isdesired, and on the area to be treated. In one aspect, administrationcan be by injection, where the composition is formulated into a liquidor gel. In other aspects, the composition can be formulated to beapplied internally to a patient. In other aspects, the composition canbe applied topically (including ophthalmically, vaginally, rectally,intranasally, orally, or directly to the skin). For example, a topicalcomposition comprising exosomes and/or microvesicles can be applied toany accessible tumor or infection, e.g. a skin tumor or other tumor(e.g., Kaposi's sarcoma); a viral infection (e.g., warts, genital warts,herpes); or a bacterial infection.

In some embodiments, the pharmaceutical composition is an injectableformulation.

The pharmaceutical composition may be administered parenterally, e.g.,intravenously, intramuscularly, intravenously, subcutaneously, orinterperitonically. The composition can be injected systemically orlocally to or near the site of a cancer. A single intravenous orintraperitoneal dose can be administered. Alternatively, a slowlong-term infusion or multiple short-term daily infusions may beutilized, typically lasting from 1 to 8 days. Alternate day or dosingonce every several days may also be utilized.

Sterile, injectable compositions are prepared by incorporating thetumoricidal and/or antimicrobial components in a suitable amount into anappropriate carrier. Suitable carriers include aqueous carriers, such aswater and aqueous buffer (e.g., phosphate buffered saline (PBS), citratebuffer, etc.), water-soluble organic solvents (e.g., polyethylene glycol300, polyethylene glycol 400, ethanol, propylene glycol, glycerin,N-methyl-2-pyrrolidone, dimethylacetamide, and dimethylsulfoxide),organic liquids/semi-solids (beeswax, d-tocopherol, oleic acid,medium-chain mono- and diglycerides), non-ionic surfactants(polyethoxylated castor oils (e.g., Cremophor EL, Cremophor RH 40,Cremophor RH 60), polysorbate 20, polysorbate 80, poloxamer 188,poloxamer 407, d-tocopherol polyethylene glycol 1000 succinate,polyethylene glycol (15)-hydroxystearate, sorbitan monooleate, oleoylpolyoxyl-6 glycerides, linoleoyl polyoxyl-6 glycerides, caprylocaproylpolyoxyl-8 glycerides, Gellucire® 44/14, Softigen® 767, and mono- anddi-fatty acid esters of PEG 300, 400, or 1750, etc.), a lipid (e.g.,castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermintoil, safflower oil, sesame oil, soybean oil, hydrogenated vegetableoils, hydrogenated soybean oil, and medium-chain triglycerides ofcoconut oil and palm seed oil), cyclodextrin (such as α-cyclodextrin,β-cyclodextrin, and γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, andsulfobutylether-β-cyclodextrin), and phospholipids (e.g.,phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,distearoylphosphatidylglycerol, 1-dimyristoylphosphatidylcholine,1-dimyristoylphosphatidylglycerol, etc.), or a mixture thereof. In someembodiments, the aqueous carrier comprises hyaluronic acid, saline,Ringer's solution, dextrose solution, Hank's solution, and other aqueousphysiologically balanced salt solutions. In some embodiments, thenonaqueous carrier comprises fixed oils, vegetable oils such as oliveoil and sesame oil, triglycerides, propylene glycol, polyethyleneglycol, or injectable organic esters such as ethyl oleate. In someembodiments, the pharmaceutically acceptable carrier further comprises aviscosity enhancing agent, such as carboxymethylcellulose or saltsthereof, sorbitol, or dextran; a substance that enhance isotonicity andchemical stability, such as phosphate buffer, bicarbonate buffer andTris buffer; a preservative such as thimerosal, cresols, formalin andbenzyl alcohol.

The injectable compositions can be in a solution or suspension, butshould be able to pass readily through an injection device such as ahollow needle. A proper viscosity may be achieved and maintained by theproper choice of solvents or excipients. In some embodiments, thepharmaceutical acceptable carrier comprises a viscosity enhancingagents. In some embodiments, the composition has a viscosity of betweenabout 5 centipoise (cP) to about 1×10⁶ cP, or about 5 cP to about 1×10⁵cP, or about 5 cP to about 1×10⁴ cP, or about 5 cP to about 1×10³ cP, orabout 6 cP to about 100 cP at 25° C.

In some embodiments, the pharmaceutical composition is an injectableextended release formulation. The tumoricidal and/or antimicrobialcomponents in the extended release formulation can be released from thecomposition to the body over an extended period of time, such as over atleast several minutes, at least one hour, at least several hours, atleast one day, at least several days, or in weeks, etc. to provide longterm and/or continuous therapeutic effect.

In some embodiments, the composition comprises a localization agentwhich allows for localized retention of the composition when deliveredto or proximate to site of a tumor, optionally for extended and/orcontinuous release of the tumoricidal and/or antimicrobial component inthe composition. Such agents include thixotropic agents, phase changingagents, such as hydrogel, bioerodible, biocompatible polymer, andcollagen gels, and the like. These compositions are in an injectable orliquid form at ambient conditions and form a viscous or gel-likebioerodible or biodegradable mass after application which limitstransport away from the site of delivery and allows for the diffusion ofthe tumoricidal and/or antimicrobial component from the composition.

The hydrogels useful in the compositions can be chemically and/orphysically cross-linked hydrogels. In situ chemical cross-linking isobtained, e.g., via photo-initiated, redox-initiated or Michael-typeaddition polymerization that preferably involve covalent bond formation.Physically cross-linked hydrogels self-assemble under external stimuliand do not rely on covalent bond formation. Temperature, pH, ionconcentration, and hydrophobic interactions are certain of the externalstimuli useful for such self-assembly and for the immobilization of suchhydrogels.

Exemplary polymers suitable for the use in the compositions includepolylactides, polyglycolides, poly(caprolactone), polyanhydrides,polyamines, polyesteramides, polyorthoesters, polydioxanones,polyacetals, polyketals, polycarbonates, polyphosphoesters,polyorthocarbonates, polyphosphazenes, succinates, poly(malic acid),poly(amino acids), polyvinylpyrrolidone, polyethylene glycol,polyhydroxycellulose, polyphosphoesters, polysaccharides, chitin,chitosan, hyaluronic acid, and copolymers, such as poloxamers,terpolymers and mixtures thereof.

In some embodiments, the localization agent is a poloxamer. Poloxamer isa nonionic triblock copolymer composed of a central hydrophobic chain ofpolyoxypropylene (e.g., (poly(propylene oxide)) flanked by twohydrophilic chains of polyoxyethylene (e.g., poly(ethylene oxide)). Inone aspect, poloxamer has the formula

HO(C₂H₄O)_(b)(C₃H₆O)_(a)(C₂H₄O)_(b)OH

wherein a is from 10 to 100, 20 to 80, 25 to 70, or 25 to 70, or from 50to 70; b is from 5 to 250, 10 to 225, 20 to 200, 50 to 200, 100 to 200,or 150 to 200. In another aspect, the poloxamer has a molecular weightfrom 2,000 to 15,000, 3,000 to 14,000, or 4,000 to 12,000. Poloxamersuseful herein are sold under the trade name Pluronic® manufactured byBASF. Non-limiting examples of poloxamers useful herein include, but arenot limited to, Pluronic® F68, P103, P105, P123, F127, and L121. Atsuitable concentrations, such as 10%-30% w/w of poloxamer, a poloxamersolution is a liquid at room temperature and forms a soft gel in thebody.

Suitable collagens include, for example, alkaline treatment of insolublecollagen extracted from various animals, or by treating with enzyme suchas pepsin, trypsin, chymotrypsin, papin or pronase. Collagen can beobtained from the skin, bone, cartilage, tendon or organs, etc. of birdsor mammals. Collagen can be flexible after curing and requires only ashort time for crosslinking, in other words, requires only a short timefor gelation. Collagen solution can also be made by dissolving collagenin a non-toxic solvent, examples of which include water, physiologicalsaline, a buffer such as borate buffer, or an aqueous solutioncontaining a salt such as sodium chloride, sodium bromide and potassiumbromide, or protein, sugar or lipid, etc.

The collagen can also form a gel even in the presence of moisture suchas that in blood or tumor, and can demonstrate a high degree ofadhesiveness with respect to living body tissue. Collagen solutions usedin the present invention can be made at various concentrations,neutralized and prepared for injection. In various embodiments, theconcentration of collagen in the composition can be at 0.2 mg/mL, 0.5mg/mL, 0.75 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL,7 mg/mL, 8 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL and 50 mg/mL,or any range between two of the numbers. Upon injection into an organ,chilled collagen gels can thermogel as they reach body temperature orabout 37° C.

In some embodiments, the pharmaceutical composition provides extended,sustained, and/or continuous release of the tumoricidal and/orantimicrobial component(s) of the composition.

Sterilisation of the composition can be done by known procedures, suchas filtration.

The final form should be stable under conditions of manufacture andstorage. Furthermore, the final pharmaceutical form should be protectedagainst contamination and should, therefore, be able to inhibit thegrowth of microorganisms such as bacteria or fungi.

Prevention or inhibition of growth of microorganisms may be achievedthrough the addition of one or more antimicrobial agents such aschlorobutanol, ascorbic acid, parabens, thimerosal, or the like. It mayalso be preferable to include agents that alter the tonicity such assugars or salts.

The pharmaceutical composition can also be made as a sterile powder.Sterile powder comprising the tumoricidal and/or antimicrobialcomponents can be prepared by methods including vacuum drying or freezedrying of a liquid composition, such as a composition comprising anaqueous carrier. The sterile powder can be reconstituted with a suitableamount of an aqueous carrier, such as PBS, to provide an injectablecomposition for administration to a patient.

In some embodiments, the compositions are formulated as a topicalcomposition applied directly to the skin for treating a skin cancer. Insome embodiments, the compositions are formulated as a topicalcomposition applied directly to other accessible cancers, for examplecervical cancer or oral cancers. In some embodiments, the topicalcomposition is applied to virus-infected cells, for example a wart. Insome embodiments, the wart is a genital (venereal) wart. In someembodiments, the wart is a common wart, flat wart, filiform/digitatewart, mosaic wart, periungal wart, or plantar wart.

Formulations for topical administration can include, emulsions, creams,aqueous solutions, oils, ointments, pastes, gels, lotions, milks, foams,suspensions and powders. In some embodiments, the pharmaceuticalcomposition is a cream or lotion. In one aspect, the topical compositioncan include one or more surfactants and/or emulsifiers. In oneembodiment, the emulsifier does not alter the structure of the exosomesand/or microvesicles. In one embodiment, the emulsifier alters thestructure of the exosomes and/or microvesicles. For example, theemulsifier may destroy the exosome and/or microvesiclestructure, therebyreleasing active factors within the exosomes and/or microvesicles.

Surfactants (or surface-active substances) that may be present areanionic, non-ionic, cationic and/or amphoteric surfactants. Typicalexamples of anionic surfactants include, but are not limited to, soaps,alkylbenzenesulfonates, alkanesulfonates, olefin sulfonates, alkyl ethersulfonates, glycerol ether sulfonates, α-methyl ester sulfonates, sulfofatty acids, alkyl sulphates, fatty alcohol ether sulphates, glycerolether sulphates, fatty acid ether sulphates, hydroxy mixed ethersulphates, monoglyceride (ether) sulphates, fatty acid amide (ether)sulphates, mono- and dialkyl sulfosuccinates, mono- and dialkylsulfosuccinamates, sulfotriglycerides, amide soaps, ether carboxylicacids and salts thereof, fatty acid isethionates, fatty acidsarcosinates, fatty acid taurides, N-acylamino acids, e.g. acyllactylates, acyl tartrates, acyl glutamates and acyl aspartates, alkyloligoglucoside sulphates, protein fatty acid condensates (in particularwheat-based vegetable products) and alkyl (ether) phosphates. Examplesof non-ionic surfactants include, but are not limited to, fatty alcoholpolyglycol ethers, alkylphenol polyglycol ethers, fatty acid polyglycolesters, fatty acid amide polyglycol ethers, fatty amine polyglycolethers, alkoxylated triglycerides, mixed ethers or mixed formals,optionally partially oxidized alk(en)yl oligoglycosides or glucoronicacid derivatives, fatty acid N-alkylglucamides, protein hydrolysates (inparticular wheat-based vegetable products), polyol fatty acid esters,sugar esters, sorbitan esters, polysorbates and amine oxides. Examplesof amphoteric or zwitterionic surfactants include, but are not limitedto, alkylbetaines, alkylamidobetaines, aminopropionates,aminoglycinates, imidazolinium-betaines and sulfobetaines.

In some embodiments, the surfactant can be fatty alcohol polyglycolether sulphates, monoglyceride sulphates, mono- and/or dialkylsulfosuccinates, fatty acid isethionates, fatty acid sarcosinates, fattyacid taurides, fatty acid glutamates, alpha-olefinsulfonates, ethercarboxylic acids, alkyl oligoglucosides, fatty acid glucamides,alkylamidobetaines, amphoacetals and/or protein fatty acid condensates.

Examples of zwitterionic surfactants include betaines, such asN-alkyl-N,N-dimethylammonium glycinates, for examplecocoalkyldimethylammonium glycinate,N-acylaminopropyl-N,N-dimethylammonium glycinates, for examplecocoacylaminopropyldimethylammonium glycinate, and2-alkyl-3-carboxymethyl-3-hydroxyethylimidazolines having in each case 8to 18 carbon atoms in the alkyl or acyl group, andcocoacylaminoethylhydroxyethyl-carboxymethyl glycinate.

In some embodiments, the emulsifier can be a nonionogenic surfactantselected from the following: the addition products of from 2 to 30 moleof ethylene oxide and/or 0 to 5 mole of propylene oxide onto linearfatty alcohols having 8 to 22 carbon atoms, onto fatty acids having 12to 22 carbon atoms, onto alkylphenols having 8 to 15 carbon atoms in thealkyl group, or onto alkylamines having 8 to 22 carbon atoms in thealkyl radical; alkyl and/or alkenyl oligoglycosides having 8 to 22carbon atoms in the alk(en)yl radical and the ethoxylated analogsthereof; the addition products of from 1 to 15 mole of ethylene oxideonto castor oil and/or hydrogenated castor oil; the addition products offrom 15 to 60 mole of ethylene oxide onto castor oil and/or hydrogenatedcastor oil; partial esters of glycerol and/or sorbitan with unsaturated,linear or saturated, branched fatty acids having 12 to 22 carbon atomsand/or hydroxycarboxylic acids having 3 to 18 carbon atoms, and theadducts thereof with 1 to 30 mole of ethylene oxide; partial esters ofpolyglycerol (average degree of self-condensation 2 to 8),trimethylolpropane, pentaerythritol, sugar alcohols (e.g. sorbitol),alkyl glucosides (e.g. methyl glucoside, butyl glucoside, laurylglucoside), and polyglucosides (e.g. cellulose) with saturated and/orunsaturated, linear or branched fatty acids having 12 to 22 carbon atomsand/or hydroxycarboxylic acids having 3 to 18 carbon atoms, and theadducts thereof with 1 to 30 mole of ethylene oxide; mixed esters ofpentaerythritol, fatty acids, citric acid and fatty alcohols and/ormixed esters of fatty acids having 6 to 22 carbon atoms, methylglucoseand polyols, preferably glycerol or polyglycerol, mono-, di- andtrialkyl phosphates, and mono-, di- and/or tri-PEG alkyl phosphates andsalts thereof; wool wax alcohols; polysiloxane-polyalkyl-polyethercopolymers and corresponding derivatives; and block copolymers, e.g.polyethylene glycol-30 dipolyhydroxystearates.

In some embodiments, the emulsifier is a polyalkylene glycol such as,for example, polyethylene glycol or polypropylene glycol. In someembodiments, the emulsifier is polyethylene glycol having a molecularweight 100 Da to 5,000 Da, 200 Da to 2,500 Da, 300 Da to 1,000 Da, 400Da to 750 Da, 550 Da to 650 Da, or about 600 Da.

In some embodiments, the emulsifier is a poloxamer as described herein.

In some embodiments, the emulsifier is composed of one or more fattyalcohols. In some embodiments, the fatty alcohol is a linear or branchedC₆ to C₃₅ fatty alcohol. Examples of fatty alcohols include, but are notlimited to, capryl alcohol (1-octanol), 2-ethyl hexanol, pelargonicalcohol (1-nonanol), capric alcohol (1-decanol, decyl alcohol), undecylalcohol (1-undecanol, undecanol, hendecanol), lauryl alcohol (dodecanol,1-dodecanol), tridecyl alcohol (1-tridecanol, tridecanol,isotridecanol), myristyl alcohol (1-tetradecanol), pentadecyl alcohol(1-pentadecanol, pentadecanol), cetyl alcohol (1-hexadecanol),palmitoleyl alcohol (cis-9-hexadecen-1-ol), heptadecyl alcohol(1-n-heptadecanol, heptadecanol), stearyl alcohol (1-octadecanol),isostearyl alcohol (16-methylheptadecan-1-ol), elaidyl alcohol(9E-octadecen-1-ol), oleyl alcohol (cis-9-octadecen-1-ol), linoleylalcohol (9Z,12Z-octadecadien-1-ol), elaidolinoleyl alcohol(9E,12E-octadecadien-1-ol), linolenyl alcohol(9Z,12Z,15Z-octadecatrien-1-ol) elaidolinolenyl alcohol(9E,12E,15-E-octadecatrien-1-ol), ricinoleyl alcohol(12-hydroxy-9-octadecen-1-ol), nonadecyl alcohol (1-nonadecanol),arachidyl alcohol (1-eicosanol), heneicosyl alcohol (1-heneicosanol),behenyl alcohol (1-docosanol), erucyl alcohol (cis-13-docosen-1-ol),lignoceryl alcohol (1-tetracosanol), ceryl alcohol (1-hexacosanol),montanyl alcohol, cluytyl alcohol (1-octacosanol), myricyl alcohol,melissyl alcohol (1-triacontanol), geddyl alcohol (1-tetratriacontanol),or cetearyl alcohol.

In some embodiments, the carrier used to produce the topical compositionis a mixture of polyethylene and one or more fatty alcohols. Forexample, the carrier comprises about 50% to about 99% by weight, about75% to about 99% by weight, about 90% to about 99% by weight, or about95% by weight polyethylene glycol and about 1% to about 50% by weight,about 1% to about 25% by weight, about 1% to about 10% by weight, orabout 5% by weight fatty alcohol. In some embodiments, the carrier is amixture of polyethylene glycol and cetyl alcohol.

The topical compositions can also include additional components suitablein such compositions. In some embodiments, the topical composition caninclude one or more of the following components: fats, waxes,pearlescent waxes, bodying agents, thickeners, superfatting agents,stabilisers, polymers, silicone compounds, lecithins, phospholipids,biogenic active ingredients, deodorants, antimicrobial agents,antiperspirants, swelling agents, insect repellents, hydrotropes,solubilizers, preservatives, perfume oils and dyes. Examples of each ofthese components are disclosed in U.S. Pat. No. 8,067,044, which isincorporated by reference with respect these components.

The topical compositions comprising the tumoricidal and/or antimicrobialcomponent(s) described herein can be prepared by mixing the component(s)with the carrier for a sufficient time such that the particles areevenly dispersed throughout the carrier. In the case when the carriercomprises two or more components, the components can be admixed with oneanother prior to the addition of the tumoricidal and/or antimicrobialcomponent. The amount of tumoricidal and/or antimicrobial componentspresent in the topical composition can vary depending upon theapplication. In some embodiments, the tumoricidal and/or antimicrobialcomponent is from 0.5% to 20%, 1% to 10%, 2% to 5%, or about 3% byweight of the topical composition.

It will be appreciated that the amounts of the tumoricidal and/orantimicrobial components in the composition in a specified case willvary according to the specific tumoricidal and/or antimicrobialcomponents being utilized, the particular compositions formulated, themode of application, and the particular situs and patient being treated.Dosages for a given host can be determined using conventionalconsiderations, e.g. by customary comparison of the differentialactivities of the composition and of a known therapy, e.g., by means ofan appropriate conventional pharmacological protocol. Physicians andformulators, skilled in the art of determining doses of pharmaceuticalagents, will have no problems determining dose according to standardrecommendations (Physician's Desk Reference, Barnhart Publishing(1999)).

Unit doses or multiple dose forms are contemplated, each offeringadvantages in certain clinical settings. The unit dose would contain apredetermined quantity of the tumoricidal and/or antimicrobialcomponents calculated to produce the desired effect(s) in the setting oftreating cancer. The multiple dose form may be particularly useful whenmultiples of single doses, or fractional doses, are required to achievethe desired ends. Either of these dosing forms may have specificationsthat are dictated by or directly dependent upon the uniquecharacteristic of the particular tumoricidal and/or antimicrobialcomponent, the particular therapeutic effect to be achieved, the diseaseto be treated, and the conditions of the patient, etc.

A unit dose will contain a therapeutically effective amount sufficientto treat a disease in a patient and may contain from about 0.001 mg to100 mg of the tumoricidal, cytotoxic, and/or antimicrobial component.The amount of the tumoricidal, cytotoxic and/or antimicrobial componentcan vary from 0.001% to 90% w/w of the composition, such as 0.001%,0.01%, 0.1%, 1%, 10%, 50%, or 90%, or in any range between any twonumbers. The disease treated may be any disease treatable by theexosomes of the invention, for example a cancer, a bacterial infection,a viral infection, or a fungal infection.

The pharmaceutical composition can further be in an oral formulationsuch as an ingestible tablet, a buccal tablet, capsule, caplet, elixir,suspension, syrup, trouche, wafer, lozenge, and the like.

The composition may be a sustained-release preparation. The compositionmay be enclosed in a hard or soft capsule, may be compressed intotablets, or may be incorporated with beverages, food or otherwise intothe diet. The percentage of the final composition and the preparationsmay, of course, be varied and may conveniently range between 1 and 90%of the weight of the final form, e.g., tablet. The amount in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

The suitable formulation of an oral composition may also contain: abinder, such as gum tragacanth, acacia, corn starch, gelatin; sweeteningagents such as lactose or sucrose; disintegrating agents such as cornstarch, alginic acid and the like; a lubricant such as magnesiumstearate; or flavoring such as peppermint, oil of wintergreen or thelike. Various other material may be present as coating or to otherwisemodify the physical form of the oral dosage unit. The oral dosage unitmay be coated with shellac, a sugar or both. Syrup or elixir may containthe tumoricidal and/or antimicrobial components, sucrose as a sweeteningagent, methyl and propylparabens as a preservative, a dye and flavoring.Any material utilized should be pharmaceutically-acceptable andsubstantially non-toxic.

Additional descriptions of preparing a pharmaceutical composition can befound in the 19th Edition of Remington's Pharmaceutical Sciences,Published by the Mack Publishing Co., Easton, Pa. 18040. The pertinentportions are incorporated herein by reference.

Method of Treatment of the Invention

The compositions described herein are useful in treating a variety ofdiseases, such as cancer. Examples of diseases that can be treated bythe compositions include malignancies of the immune system, thelymphatic system, and the hematopoietic system, formed tumors, and solidtumors. Non-limiting examples of cancers that can be treated with thecompositions include mast cell leukemia, acute myelogenous leukemia(AML), erythroleukemia, myeloid disorders (e.g., myeloid leukemia,multiple myeloma, and erythroleukemia), germ cell tumors, lungcarcinoma, small-cell lung carcinoma, gastrointestinal stromal tumors,neuroblastoma, cervical carcinoma, ovarian carcinoma, brain carcinoma,breast carcinoma, ovary carcinoma, endometrium carcinoma, kidneycarcinoma, thyroid carcinoma, bladder carcinoma, colon carcinoma,pancreas carcinoma and prostate carcinoma, skin carcinoma such asmelanoma, adenomas (e.g., villous colon adenoma), and sarcomas (e.g.,osteosarcoma), etc.

In some embodiments, the pharmaceutical compositions described hereinare used to treat skin cancer, for example melanoma, squamous cellcancer, basal cell cancer, or mast cell tumors.

In some embodiments, the pharmaceutical compositions described hereinare used to treat other epithelial cell cancers, for example cervicalcancer or oral cancer. In some embodiments, the pharmaceuticalcompositions described herein are used to treat virus-infected cells,for example warts.

In some embodiments, the pharmaceutical compositions described hereinare used to modulate the immune system. For example, exosomes and/ormicrovesicles may induce cytotoxic T cell response (e.g., againsttumors), and/or induce apoptosis (e.g., of activated immune cells).Exosomes and/or microvesicles may also play a role in immunesurveillance.

In some embodiments, the pharmaceutical compositions described hereinare useful in treating infections. In one embodiment, the pharmaceuticalcompositions described herein are useful in treating infections bypathogenic viruses. Pathogenic viruses include, without limitation,human papillomavirus, human immunodeficiency virus, Epstein-Barr virus,cytomegalovirus, Ebola virus, Marburg virus, influenza, respiratorysyncytial virus, poxvirus, varicella-zoster virus, and herpes. In oneembodiment, the pharmaceutical compositions described herein are usefulin treating bacterial infections. Infectious bacteria include, withoutlimitation, Streptococcus, Staphylococcus, Cryptococcus, Chlamydia,Escherichia, Pseudomonas, Clostridium, and Candida, includingantibiotic-resistant strains of any of the above. In one embodiment, thepharmaceutical compositions described herein are useful in treatinginfections caused by other microbes, including fungus and yeast.

With mammals, including humans and domesticated animals, the effectiveamount can be administered on the basis of body surface area to becovered (e.g., affected area), for example in a topical composition. Asuitable dose range is from about 0.001 mg to about 100 mg of equivalentper m² body surface area of a tumoricidal and/or antimicrobialcomponent, for instance from about 0.005 mg/m² to about 50 mg/m². Thedosage can be administered daily, such as once, twice, three times ormore per day, or every two or several days, or every week, etc. Thefrequency of administration can be reduced if an extended releaseformulation is administered.

In one embodiment, the effective amount can be administered on the basisof tumor volume. A suitable dose range is from about 1:100 to about1:10,000 exosome/microvesicle preparation to tumor volume ratio. In oneembodiment, the suitable dose range from about 1:100 to about 1:1,000exosome/microvesicle preparation to tumor volume ratio. In oneembodiment, the suitable dose range from about 1:1,000 to about 1:10,000exosome/microvesicle preparation to tumor volume ratio.

In one embodiment, the effective amount can be administered based onpatient body weight. A suitable dose range is from about 1 μg to about100 mg exosome/microvesicle preparation per kg body weight. In oneembodiment, the suitable dose range from about 1 μg to about 10 mgexosome/microvesicle preparation per kg body weight. In one embodiment,the suitable dose range from about 1 μg to about 100 μgexosome/microvesicle preparation per kg body weight. In one embodiment,the suitable dose range from about 10 μg to about 10 mgexosome/microvesicle preparation per kg body weight. In one embodiment,the suitable dose range from about 100 μg to about 10 mgexosome/microvesicle preparation per kg body weight.

The dosage and frequency of administration may depend on the type offormulation, the disease being treated, the amount of the tumoricidaland/or antimicrobial component, the patient's age, gender, species,other conditions, etc.

Combination Therapy

In another aspect, the compositions described herein can be administeredin conjunction with other cancer therapies, such as surgery, radiation,chemotherapy (e.g., cisplatin, carboplatin, oxaliplatin, satraplatin,and picoplatin, especially cisplatin and carboplatin; taxanes, such aspaclitaxel and docetaxel; and anthracyclines such as daunorubicin,doxorubicin, epirubicin, idarubicin, or valrubicin, etc.), cell basedtherapy (e.g., NK-92 cell therapy), antibody therapy, etc. In someembodiments, the composition can be administered in conjunction with oneor more cytokines as described herein.

The following examples are included to illustrate the invention and notto limit the invention. All publications or references cited in thepresent specification are hereby incorporated by reference.

EXAMPLES Example 1: Antimicrobial Effect of NK-92 Cells

NK-92 cells were incubated with Cryptococcus neoformans in round-bottomplates in Myelocult medium for 24 hours. An effector:target (NK-92: C.neoformans) ratio of 100:1 (1×10⁶:1×10⁴ cells/well) was used. C.neoformans (1×10⁴ cells/well) were incubated in Myelocult medium withoutNK-92 cells as a control. After the 24 hour incubation, the cultureswere serially diluted and plated on Sabouraud agar plates. Plates wereincubated at room temperature for 48 hours. The number of colonies ateach dilution were measured.

The number of colonies per plate at each dilution are indicated inFIG. 1. White bars: NK-92 cells+C. neoformans. Black bars: C. neoformansalone. FIG. 2 indicates that NK-92 cells inhibited C. neoformans growthby approximately 60%.

Similar results were observed when NK-92 cells were incubated withanother species of fungus (a species of Aspergillus). NK-92 cells causedhyphal damage to the fungus in a dose- and time-dependent manner.

Example 2: Isolation of Exosomes and/or Microvesicles from NK-92 CellSupernatant

Extracellular vesicles (exosomes and/or microvesicles) were isolated byultracentrifugation. Cultured NK-92 cells were centrifuged at 300×g for10 minutes, and the cell pellet discarded. The supernatant wascentrifuged at 2000×g for 20 minutes, and the pellet (cell debris) wasdiscarded. The resulting supernatant was subjected toultracentrifugation at 100,000×g for 80 minutes. The resulting pelletwas washed with phosphate-buffered saline and subjected toultracentrifugation at 100,000×g for 80 minutes. The washed pelletcontaining exosomes and microvesicles (EV/MV preparation) was retainedfor further studies.

Example 3: Characterization of Extracellular Vesicles

NK-92 cells were cultured under a variety of conditions, and theextracellular vesicles were isolated from the media as described inExample 2. The culture conditions are provided in Table 1. Cells wereconditioned in exofree FBS for at least 24 to 46 hours prior toharvesting of exosomes. ExoFree FBS is exosome depleted to avoidcontamination of the NK-92 extracellular vesicle preparations.

TABLE 1 NK-92 Growth Conditions Number of Media ExoFree IL-2 Lane cellsBackbone FBS (%) (IU/mL) 4 4.0 × 10⁷ αMEM(NK) 5 500 5 4.8 × 10⁷ RPMI 10500 6 4.0 × 10⁷ X-Vivo10¹ 5 500 7 3.6 × 10⁷ X-Vivo10¹ 0 500 ¹Lonza GroupLtd.

EV/MV preparations were analyzed for the presence of several proteinsusing standard Western blotting techniques, as shown in FIG. 3A and FIG.3B. The MCF-7 cell line was used as a positive control for exosomeproduction and a negative control for cytolytic proteins. NK-92 cellpellet (NK-92 cells) were used as a positive control. MCF-7 cells werecultured in DMEM with 10% ExoFree fetal bovine serum (FBS) and 2 mML-Glutamine.

Extracellular vesicles from both NK-92 and MCF-7 cells were positive forRab5B, an exosome marker. NK-92 EV/MV preparations (but not MCF-7 EV/MVpreparations) were also positive for several apoptosis-inducing and/orcytolytic proteins that are known to be involved in NK cell activity,including perforin, Fas ligand (FasL), granzyme B, and granulysin.However, the amount of each of these proteins appears to be dependant onNK-92 cell growth conditions, including media backbone (e.g., αMEM(NK),RPMI, X-Vivo) and serum concentration.

EV/MV preparations from NK-92 cells were analyzed for contamination byother organelles. EV/MV isolated from NK-92 show some contamination withnuclear material (small apoptotic bodies) but were free of othercontaminating organelles, as shown in FIG. 3B.

Example 4: Cytotoxicity of EV/MV Preparations

The cytotoxicity of EV/MV preparations against Jurkat cells was testedusing the culture conditions indicated in Table 1. Jurkat cells (2×10⁴)were incubated in 170 μL medium (with ExoFree FBS) and 5, 15, or 30 μLof EV/MV preparation (with PBS to achieve total incubation volume of 200μL) for 2 hours or 20 hours. Cytotoxicity was determined by propidiumiodide (PI) assay, and data are expressed as the percent of cells thatare PI positive (indicative of dead cells).

As shown in FIG. 4, EV/MV preparations from NK-92 cells, but not MCF-7cells, have cytotoxic activity against Jurkat cells in vitro. Mediacomposition changes the lytic potential of EV/MV, and the presence ofserum in the growth medium stimulates production of lytic EV/MV by NK-92cells.

1.-10. (canceled)
 11. A production intermediate for a cell-basedproduct, comprising: a plurality of NK-92 cells in a culture mediumcomprising a cytokine; wherein the NK-92 cells are a subpopulation ofwild type NK-92 cells or modified NK-92 cells; wherein the subpopulationof wild type NK-92 cells or modified NK-92 cells produces cytotoxicexosomes when grown in the culture medium; and wherein the culturemedium comprises exosomes and/or microvesicles that were produced by thesubpopulation of wild type NK-92 cells or modified NK-92 cells in theculture medium.
 12. The intermediate of claim 1, wherein the cytokine isIL-2, wherein the medium is a serum-free medium, and/or wherein themedium is supplemented with L-asparagine, L-glutamine, L-serine,interferon gamma, and/or IL-15.
 13. The intermediate of claim 1, whereinthe subpopulation of the wild type NK-92 cells or the modified NK-92cells express different surface markers or proteins as compared to NK-92cells having ATCC deposit number CRL-2407.
 14. The intermediate of claim1, wherein the subpopulation of the wild type NK-92 cells or themodified NK-92 cells are isolated.
 15. The intermediate of claim 1,wherein the subpopulation of the wild type NK-92 cells or the modifiedNK-92 cells expresses an Fc receptor selected from the group consistingof an Fcγ receptor, CD16 (FcγRIII-A), FCγRI (CD64), FCγRII (CD32),FCγRIII, FcRn, Fcα, or Fcε.
 16. The intermediate of claim 1, wherein thesubpopulation of the wild type NK-92 cells or the modified NK-92 cellsthe NK-92 cells comprises NK-92-CD16, NK-92-CD16-γ, or NK-92-CD16-ζ. 17.The intermediate of claim 1, wherein the subpopulation of the wild typeNK-92 cells or the modified NK-92 cells expresses IL-2, an IL-2receptor, IL-15, an IL-15 receptor, IL-18, an IL-18 receptor, IL-21,and/or an IL-21 receptor.
 18. A method of producing a cell-basedproduct, comprising: growing a plurality of NK-92 cells in a culturemedium comprising a cytokine; wherein the NK-92 cells are asubpopulation of wild type NK-92 cells or modified NK-92 cells; whereinthe subpopulation of wild type NK-92 cells or modified NK-92 cells isgrown under conditions and for a time sufficient to produce cytotoxicexosomes; and removing at least some of the culture medium containingthe exosomes and/or microvesicles from the subpopulation of wild typeNK-92 cells or modified NK-92 cells in the culture medium.
 19. Themethod of claim 8, wherein the cytokine is IL-2.
 20. The method of claim8, wherein the medium is a serum-free medium.
 21. The method of claim 8,wherein the medium is supplemented with L-asparagine, L-glutamine,L-serine, interferon gamma, and/or IL-15.
 22. The method of claim 8,wherein the subpopulation of the wild type NK-92 cells or the modifiedNK-92 cells express an Fc receptor selected from the group consisting ofan Fcγ receptor, CD16 (FcγRIII-A), FCγRI (CD64), FCγRII (CD32), FCγRIII,FcRn, Fcα, or Fcε.
 23. The method of claim 8, wherein the subpopulationof the wild type NK-92 cells or the modified NK-92 cells the NK-92 cellscomprise NK-92-CD16, NK-92-CD16-γ, or NK-92-CD16-ζ.
 24. The method ofclaim 8, wherein the subpopulation of the wild type NK-92 cells or themodified NK-92 cells expresses IL-2, an IL-2 receptor, IL-15, an IL-15receptor, IL-18, an IL-18 receptor, IL-21, and/or an IL-21 receptor. 25.A cell-based method of treating cancer, comprising: administering to apatient in need thereof a composition that comprises a plurality ofNK-92 cells and cytotoxic exosomes and/or microvesicles generated fromthe NK-92 cells; wherein the NK-92 cells are a subpopulation of wildtype NK-92 cells or modified NK-92 cells; wherein the subpopulation ofwild type NK-92 cells or modified NK-92 cells were grown underconditions and for a time sufficient to produce the cytotoxic exosomes.26. The method of claim 15, wherein the composition is administered tothe patient in conjunction with surgery, radiation, chemotherapy, and/orantibody therapy.
 27. The method of claim 15, wherein the composition isadministered to the patient with one or more cytokines.
 28. The methodof claim 15, wherein the composition is administered to the patient byinfusion or injection.
 29. The method of claim 15, wherein thecomposition is administered to the patient to induce a cytotoxic T cellresponse and/or induce apoptosis.
 30. The method of claim 15, whereinthe cancer is mast cell leukemia, acute myelogenous leukemia (AML),erythroleukemia, a myeloid cancer, a germ cell tumor, lung carcinoma,small-cell lung carcinoma, a gastrointestinal stromal tumor,neuroblastoma, cervical carcinoma, ovarian carcinoma, brain carcinoma,breast carcinoma, ovary carcinoma, endometrium carcinoma, kidneycarcinoma, thyroid carcinoma, bladder carcinoma, colon carcinoma,pancreas carcinoma, prostate carcinoma, melanoma, an adenoma, or asarcoma.